Transcriptome transfer produces cellular phenotype conversion

ABSTRACT

The present invention includes methods for effecting phenotype conversion in a cell by transfecting the cell with phenotype-converting nucleic acid. Expression of the nucleic acids results in a phenotype conversion in the transfected cell. Preferably the phenotype-converting nucleic acid is a transcriptome, and more preferably an mRNA transcriptome.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in part of U.S. patent applicationSer. No. 12/755,277, filed on Apr. 6, 2010 which is acontinuation-in-part of U.S. application Ser. No. 12/086,471, filed onJun. 13, 2008, which is the National Stage application of PCTInternational Application No. PCT/US2006/047480, filed on Dec. 12, 2006,which in turn claims the benefit pursuant to 35 U.S.C. §119(e) of U.S.Provisional Application No. 60/749,941, filed on Dec. 13, 2005; and alsoclaims the benefit pursuant to 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 61/167,286, filed on Apr. 7, 2009, each of which ishereby incorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported in part by funds obtained from the U.S.Government (National Institutes of Health Director's Pioneer Awardnumber DP 1-OD-04117), and the U.S. Government therefore has certainrights in the invention.

BACKGROUND OF THE INVENTION

Cellular phenotype is the conglomerate of multiple cellular processesinvolving gene and protein expression that result in the elaboration ofa cell's particular morphology and function. It has been thought thatdifferentiated post-mitotic cells have their genomes hard wired withlittle ability for phenotypic plasticity. Emerging evidence has,however, demonstrated the reversibility and flexibility of the cellularphenotype. It has been shown that fertile adult male and female frogscan be obtained by injecting endoderm nuclei into enucleated eggs(Gurdon J B, Elsdale T R, & Fischberg M (1958) sexually matureindividuals of Xenopus laevis from the transplantation of single somaticnuclei. Nature 182:64-65). This result not only forms the foundation ofthe field in nuclear transplantation, but also provides evidence thatthe cytoplasmic components of a differentiated cell can support nuclearreprogramming. Generation of induced pluripotent stem (iPS) cells bytransfection transcription factors into dividing fibroblasts (TakahashiK & Yamanaka S (2006) Induction of pluripotent stem cells from mouseembryonic and adult fibroblast cultures by defined factors. Cell126:663-676), followed by cell selection represent a new strategy toglobally revert a mature cell into a different cell type. See: HuangfuD, et al. (2008) Induction of pluripotent stem cells from primary humanfibroblasts with only Oct4 and Sox2. Nat Biotechnol 26:1269-1275; Kim JB, et al. (2008) Pluripotent stem cells induced from adult neural stemcells by reprogramming with two factors. Nature 454:646-650; Nakagawa M,et al. (2008) Generation of induced pluripotent stem cells without Mycfrom mouse and human fibroblasts. Nat Biotechnol 26:101-106; Maherali N,et al. (2007) Directly reprogrammed fibroblasts show global epigeneticremodeling and widespread tissue contribution. Cell Stem Cell 1:55-70;Okita K, Ichisaka T, & Yamanaka S (2007) Generation ofgermline-competent induced pluripotent stem cells. Nature 448:313-317;and Stadtfeld M, Nagaya M, Utikal J, Weir G, & Hochedlinger K (2008)Induced Pluripotent Stem Cells Generated Without Viral Integration.Science 322:945-949. The need for re-differentiation of theseES-like-iPS cells into desired cell types, however, adds a layer ofcomplexity that is difficult to control (Wernig M, et al. (2008) Neuronsderived from reprogrammed fibroblasts functionally integrate into thefetal brain and improve symptoms of rats with Parkinson's disease. ProcNatl Acad Sci USA 105:5856-5861; Hanna 3, et al. (2007) Treatment ofsickle cell anemia mouse model with iPS cells generated from autologousskin. Science 318:1920-1923). Nevertheless, studies of nuclearreprogramming from genomic and epigenetic modification, as seen fromsomatic-cell-nuclear-transfer-cloned animals and iPS cells, suggests theflexibility of a differentiated phenotype as well as the dynamic changesof a genome (Maherali N, et al. (2007) Directly reprogrammed fibroblastsshow global epigenetic remodeling and widespread tissue contribution.Cell Stem Cell 1:55-70).

Cardiomyocytes are one of the most sought after cells in regenerativemedicine because of their role in repairing injured heart by replacingthe lost tissue (Germani et al., 2007, Trends Mol Med 13(3): 125-33).Functional cardiomyocyte-like cells have been generated from embryonicstem cell, induced pluripotent stem (iPS) cell and direct conversion offibroblast using defined transcription factor transduction (Takeuchi etal., 2009, Nature 459(7247): 708-11; Boheler et al., 2002, Circ Res91(3): 189-201; Zhang et al., 2009, Circ Res 104(4): e30-41; Ieda etal., 2010, Cell 142(3): 375-86). However, carcinogenesis and earlysenescence often develop in transcription factors induced cells(Knoepfler, 2009, Stem Cells 27(5): 1050-6; Feng et al., 2010, StemCells 28(4): 704-12). It has been shown previously that transfer of thetranscriptome (TIPeR) from rat astrocyte into rat neuron converted theneuron into an astrocyte-like cell (Sul et al., 2009, Proc Natl Acad SciUSA 106(18): 7624-9).

Despite the development and refinement of the techniques discussedabove, there remains a need in the art for methods and compositions foreffecting phenotypic change in a cell. This invention addresses thatneed.

BRIEF SUMMARY OF THE INVENTION

The present invention encompasses a method of effecting phenotypeconversion in a cell. The method comprises introducing a second cell,the recipient cell, having a particular phenotype withphenotype-converting nucleic acid from a first cell, the donor cell,having a particular phenotype, wherein the phenotype of the first cellis different from that of the second cell. In one embodiment, thenucleic acid is introduced to the second cell by transfection. In someembodiments, the first cell is pre-treated with a transcriptioninhibitor. In some embodiments, the second cell is pre-treated with atranscription inhibitor before it is transfected. In some embodiments,the phenotype of the first cell differs from the phenotype of the secondcell by one or more of: species, tissue type, differentiation degree,disease state, exposure to a toxin, exposure to a pathogen, and exposureto a candidate therapeutic. Optionally, the method further comprisestransfecting the second cell at least a second time with the first cellmRNA transcriptome.

In one embodiment, the first cell is a cardiomyocyte. In someembodiments, the second cell is a fibroblast. Preferably, thephenotype-converting nucleic acid is the transcriptome and morepreferably the mRNA transcriptome of the first cell. In one embodiment,the mRNA transcriptome comprises mRNA transcripts having an average sizebetween about 1 kb to about 5 kb.

In some embodiments, the phenotype-converting nucleic acid furthercomprises one or more exogenous nucleic acids selected from the groupconsisting of mRNA, siRNA, miRNA, hnRNA, tRNA, non-coding RNA andcombinations thereof.

In some embodiments, the cell is selected from the group consisting of aeukaryotic cell and a prokaryotic cell. The eukaryotic cell can be anon-mammalian cell or it can be a mammalian cell. In some embodiments,the eukaryotic cell is a human cell.

In some embodiments, phenotype conversion comprises a change in one ormore of gene expression, protein expression, immunological markers,morphology, physiology, synthesis of bioproducts, and membrane lipidcomposition. In some embodiments, phenotype conversion comprises achange in expression of at least 100 genes. Phenotype conversion cancomprise up-regulation of genes associated with chromosomal remodeling.In some embodiments, at least about 5% of differentially expressed genesin the second cell change expression to a level observed for the firstcell.

In some embodiments, phenotype conversion persists for at least 2 weeks.In other embodiments, phenotype conversion persists for the lifetime ofthe cell.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 depicts the results of experiments showing that tCardiomyocytesdisplay cardiomyocyte-like morphology. (Top, photomicrographs) LivetCardiomyocyte cells display triangular or elongated morphologies(Upper), but fibro-TIPeR cells have an enlarged flat shape similar tofibroblast cells (Lower). (Bottom graph) The cell length-to-width ratioof cardio-TIPeR cells shows that the subpopulation of cardio-TIPeR cellsdiffers from fibroblast and fibro-TIPeR cells but is similar to adultventricular myocytes.

FIG. 2 depicts the results of experiments demonstrating thattCardiomyocytes express cardiac antigenic markers continuously.tCardiomyocytes and fibro-TIPeR cells are double-immunostained withanticardiac troponin I antibody (left column) and anti-Nkx2.5 antibody(middle column). tCardiomyocytes show similar expression patterns ofcardiac antigenic markers as adult ventricular myocytes from 2 wk to 8wk of incubation. (Insets) Immunostaining results of correspondingfibro-TIPeR cells.

FIG. 3, comprising FIGS. 3A and 3B, depicts the results of experimentsdemonstrating that global gene expression of tCardiomyocytes isreprogrammed toward adult ventricular myocytes. (3A) Dendrogram showinghierarchical clustering (Euclidean distance, complete linkage) of singleAVMs, fibroblasts, cardio-TIPeR, and fibro-TIPeR using the expressionvalues of 3,257 informative genes. Bootstrap values from 1,000 timesresampling are shown. (3B) Heat map showing cardiomyocyte-specific genesthat are up-regulated in the tCardiomyocytes (n=262) andfibroblast-specific genes that are downregulated in the tCardiomyocytes(n=136). Relevantly enriched Gene Ontology categories are annotated.

FIG. 4, comprising FIGS. 4A and 4B, depicts the results of experimentsmeasuring the electrical properties of tCardiomyocytes. tCardiomyocytesgain electrical functions similar to adult ventricular myocytes. (4A)tCardiomyocyte expresses stereotypical cardiac action potential (firstrow). Other tCardiomyocytes display a more diverse pattern of actionpotential profiles. (Inset) Patch clamp results of fibroblasts and adultventricular myocytes. (4B) tCardiomyocytes show intracellular local Ca2+oscillations. Local areas are selected (white dashed circles) and localCa2+ changes are recorded (line graphs) by time course.

FIG. 5, comprising FIGS. 5A and 5B, depicts the results of experimentsdemonstrating that tCardiomyocytes generated from mouse astrocytes showphenotypic characteristics of AVMs. (5A) Astrocyte-generatedtCardiomyocytes (3 wk after the transfection) double-immunostained withanti-troponin I antibody (Top) and anti-Nkx2.5 antibody (Middle). Nucleiare shown at bottom. (Insets) Immunostaining results of correspondingmock-transfected astrocytes. (5B) Dendrogram and heat map showinghierarchical clustering (Pearson distance, complete method) of singleAVMs, astrocyte-generated tCardiomyocytes, and astrocytes (4 wk afterthe transfection). Differentially expressed 1,690 informative genes areselected by P value cutoff (P<0.01). Bootstrap values (%) from 1,000resamplings are shown at each node.

DETAILED DESCRIPTION OF THE INVENTION

cDNA microarray analysis has shown that phenotypic differences at thecellular level are associated with differences in the presence, absenceand abundances of particular RNAs. The invention described herein arisesfrom the discovery that the relative abundances of RNAs within apopulation themselves can elaborate cellular phenotype. Specifically,the invention provides a method of effecting a phenotype conversion inrecipient cell by introducing phenotype-converting nucleic acid from adonor cell into the recipient cell. In a preferred embodiment, thephenotype-converting nucleic acid is the mRNA transcriptome of the donorcell. The discovery described herein indicates that the plasticity ofthe non-dividing genome is much greater than previously imagined.

Phenotype-converting nucleic acid may include, without limitation, mRNA,siRNA, microRNA, tRNA, hnRNA, total RNA, DNA, and combinations thereof,such that the introduction of these nucleic acids into a cell and thesubsequent expression of these nucleic acids results in a combinedphenotype due to the multiple expression of these nucleic acids andtheir interactions with each other. Unlike expression systems known inthe art, where one or only a few nucleic acids are expressed, themethods of the present invention permit the expression of multiplenucleic acids essentially simultaneously, resulting in an expressionsystem closely mirroring the interaction of various nucleic acids andtheir expression products in a natural environment. Thus, the presentinvention permits the introduction of a complex mixture of nucleic acidsinto a cell to produce a multigenic effect, thereby effecting phenotypeconversion of a cell.

The methods of the present invention are performed by transfecting amixture of nucleic acids into live cells. The methods of the inventionutilize a wide variety of methods of transfection, including those knownin the art and those described herein.

The present invention permits the transfection of nucleic acid,preferably mRNA and/or DNA into a cell with accurate control of theamount of nucleic acid entering the cell, thus allowing the skilledartisan to mimic the expression level of nucleic acid in a cell underdesired conditions, as disclosed elsewhere herein. That is, the presentinvention allows the skilled artisan to accurately control the level ofnucleic acid transfected into a cell by modulating the concentration ofnucleic acid in the extracellular environment of the cell.

In one embodiment, the present invention includes methods for phenotypeconversion of a cell using laser-aided poration of live cell membranescoupled with bath application of nucleic acids, preferably atranscriptome, in order to transfect a mixture of nucleic acids into alive cell. Photoporation is advantageous in enabling highlylocation-specific transfection of a cell and permitting multipleporation events, while not detrimental to cellular function orviability. Further, the precise amount of nucleic acid transfected intoa cell can be modulated through regulation of laser intensity, pore sizeand number, and duration of membrane opening, as well as repetition oftransfection.

The methods of the present invention are not limited to cells, but canfurther include live slices of tissue and live animals, preferablymammals, as disclosed elsewhere herein. The methods of the presentinvention can further comprise other non-mammalian cells eukaryoticcells and prokaryotic cells, such as bacterial cells, yeast cells, plantcells, protozoa, insect cells, fungal cells, including filamentous andnon-filamentous fungi, and the like.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, “phenotype conversion” refers to the induction orestablishment of a destination phenotype. Phenotype conversion comprisesa change in at least one of gene expression, protein expression,immunological markers, morphology, physiology, synthesis of bioproducts(e.g., dopamine) and membrane lipid composition.

As used herein, a “destination phenotype” refers to a phenotype ofinterest that is induced in a recipient cell by the introduction thereinof a mixture of nucleic acids. The phenotype of interest may be anyphenotype. For example, a destination phenotype may be a morphologicalchange. A destination phenotype may be a physiological change, such asthe presence of voltage-gated calcium receptors in a recipient cell. Adestination phenotype may comprise more than one phenotypic change andmay even cause the cell to assume characteristics of a different tissuetype from its original tissue type.

The phrase “phenotype-converting nucleic acid” refers herein to amixture of nucleic acid that is capable of establishing a destinationphenotype in a recipient cell. Phenotype-converting nucleic acid is notlimited to the empirical content of RNA in a donor cell, but rather,encompasses the relative abundance of each RNA with respect to each in apopulation of RNAs such that the population of RNAs are necessary andsufficient to induce a destination phenotype in a recipient cell.

As used herein, “transcriptome” refers to the collection of all genetranscripts in a given cell and comprises both coding RNA (mRNAs) andnon-coding RNAs (e.g., siRNA, miRNA, hnRNA, tRNA, etc.). As used herein,an “mRNA transcriptome” refers to the population of all mRNA moleculespresent (in the appropriate relative abundances) in a given cell. AnmRNA transcriptome comprises the transcripts that encode the proteinsnecessary to generate and maintain the phenotype of the cell. As usedherein, an mRNA transcriptome may or may not further comprise mRNAmolecules that encode proteins for general cell existence, e.g.,housekeeping genes and the like.

As used herein, “TIPeR” refers to the process of transfecting arecipient cell with a transcriptome from a donor cell. A cell that hasundergone this process may be referred to herein as a TiPeRed cell.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated,then the animal's health continues to deteriorate. In contrast, a“disorder” in an animal is a state of health in which the animal is ableto maintain homeostasis, but in which the animal's state of health isless favorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

A “fluid medium” or “fluid media” is used herein to refer to a form ofmatter, such as air, liquid, solid or plasma, preferably liquid, that iscapable of flowing.

An “isolated cell” refers to a cell which has been separated from othercomponents and/or cells which naturally accompany the isolated cell in atissue or mammal.

As applied to a protein, a “fragment” of a polypeptide, protein or anantigen, is about 6 amino acids in length. More preferably, the fragmentof a protein is about 8 amino acids, even more preferably, at leastabout 10, yet more preferably, at least about 15, even more preferably,at least about 20, yet more preferably, at least about 30, even morepreferably, about 40, and more preferably, at least about 50, morepreferably, at least about 60, yet more preferably, at least about 70,even more preferably, at least about 80, and more preferably, at leastabout 100 amino acids in length amino acids in length, and any and allintegers there between.

A “genomic DNA” is a DNA strand which has a nucleotide sequencehomologous with a gene as it exists in the natural host. By way ofexample, a fragment of a chromosome is a genomic DNA.

As used herein, an “inhibitory nucleic acid” refers to an siRNA, amicroRNA, an antisense nucleic acid or a ribozyme.

As used herein, “locally transfecting” a nucleic acid refers tointroducing a nucleic acid into a region of cytoplasm that is not theentirety of the cytoplasm of a cell optionally comprising a cellularprocess.

As used herein, “porate” or “porates” refers to creating a hole in asurface through which compounds can pass.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arecompletely or 100% homologous at that position. The percent homologybetween two sequences is a direct function of the number of matching orhomologous positions, e.g., if half (e.g., five positions in a polymerten subunits in length) of the positions in two compound sequences arehomologous then the two sequences are 50% identical, if 90% of thepositions, e.g., 9 of 10, are matched or homologous, the two sequencesshare 90% homology. By way of example, the DNA sequences 5′ATTGCC3′ and5′TATGGC3′ share 50% homology.

In addition, when the terms “homology” or “identity” are used herein torefer to the nucleic acids and proteins, it should be construed to beapplied to homology or identity at both the nucleic acid and the aminoacid sequence levels.

The term “multigenic phenotype” is used herein to refer to a phenotypein a cell, tissue or animal that is mediated by the expression or lackof expression of two or more nucleic acids encoding a protein, whereinthe nucleic acids are exogenously provided to the cell, tissue oranimal.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell.

A recombinant polynucleotide may serve a non-coding function (e.g.,promoter, origin of replication, ribosome-binding site, etc.) as well.

A host cell that comprises a recombinant polynucleotide is referred toas a “recombinant host cell.” A gene which is expressed in a recombinanthost cell wherein the gene comprises a recombinant polynucleotide,produces a “recombinant polypeptide.”

A “recombinant polypeptide” is one which is produced upon expression ofa recombinant polynucleotide.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides.

The term “peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

“Phototransfection” is used herein to refer to a process by which a holeis created in a barrier, such as a cell membrane, using a photon source,such as a laser, and two or more nucleic acids, wherein the nucleicacids encode different polypeptides, are inserted into a cell throughthe hole in the cell membrane.

By “tag” polypeptide is meant any protein which, when linked by apeptide bond to a protein of interest, may be used to localize theprotein, to purify it from a cell extract, to immobilize it for use inbinding assays, or to otherwise study its biological properties and/orfunction.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention provides methods of introducing mixtures ofnucleic acids into a recipient cell to produce a phenotype-conversion inthe recipient cell. The present invention comprises transfectingphenotype-converting nucleic acid, preferably RNA and/or DNA, even morepreferably mRNA, and most preferably, an mRNA transcriptome, locallyinto a recipient cell. The phenotype of the donor cell is different fromthe phenotype of the recipient cell. The difference in phenotype may beany difference, such a difference in species, tissue type, extent ofdifferentiation, exposure to a drug or pathogen, disease state, growthconditions and so forth, wherein the difference is known or suspected ofresulting from a difference in gene expression.

As shown herein, transfection with an mRNA transcriptome yields a highdegree of phenotype conversion. Where multiple cells are transfected inaccordance with the method of the invention, at least about 25% of thecells undergo phenotype conversion. In some embodiments, phenotypeconversion in at least about 35% of recipient cells is observed.

The recipient cell may be any type of cell. A recipient cell may be aeukaryotic cell or a prokaryotic cell. When the cell is a eukaryoticcell, the cell is preferably a mammalian cell, including but not limitedto human, non-human primate, mouse, rabbit, rat, goat, guinea pig, horsecell, and the like. A non-mammalian eukaryotic cell includes a yeastcell, a plant cell, an insect cell, a protozoan cell and a fungal cell,including filamentous and non-filamentous fungi. When the cell is aprokaryotic cell, the cell is a bacterial cell. A recipient cell may bea differentiated cell and/or a non-dividing cell. The cell may also be aprogenitor cell or a stein cell. Preferably, the recipient cell is atissue-specific cell, more preferably a mammalian tissue-specific celland more preferably still, a human tissue-specific cell. Non-limitingexamples of cells suitable as recipient cells include epithelial cells,neurons, fibroblasts, embryonic fibroblasts, keratinocytes, adult stemcells, embryonic stem cells, and cardiomyocytes.

To obtain the desired phenotype conversion, recipient cells arepreferably phenotypically-pliable cells. Phenotypically-pliable cellsare cells whose phenotype is amenable to changing under the condition'sof the method of the invention. Non-limiting examples ofphenotypically-pliable cells include neurons, fibroblasts, embryonicfibroblasts, adult stem cells and embryonic stem cells. Preferably, thecell is a fibroblast, and the nucleic acid is RNA, even more preferably,mRNA and more preferably still, an mRNA transcriptome.

In the method of the invention, nucleic acid is transferred into a cellto initiate phenotype conversion in the recipient cell. As used herein,phenotype conversion comprises a change in at least one of geneexpression, protein expression, immunological markers, morphology,physiology, synthesis of bioproducts (e.g., dopamine) and membrane lipidcomposition. Preferably, the change yields a phenotype associated withor indicative of the cell from which the transfected RNA or DNA isobtained. Preferably, phenotype conversion in the recipient cellcomprises two or more changes. More preferably, phenotype conversioncomprises three or more changes. In one embodiment, phenotype conversioncomprises a change in physiology. In another embodiment, phenotypeconversion comprises a change in morphology and a change in physiologyof the recipient cell. Phenotype conversion may be accompanied bychanges in expression in hundreds of genes. For instance, expression ofgenes quiescent in both the donor and the recipient cells may be de novoup-regulated. Genes associated with chromosomal remodeling, such asgenes involved in chromosome and DNA metabolism related process, may beup-regulated in cells having phenotype conversion. Genes annotated “BP”in the Gene Ontology (“GO”) database are considered associated withchromosomal remodeling (The Gene Ontology Consortium (2000) “Geneontology: tool for the unification of biology,” Nature Genet. 25:25-29).The GO database is publicly available (see www.geneontology.org). Insome embodiments, at least about 5%, more preferably about 7%, 10%, 15%and more preferably still at least about 25% of genes that are expresseddifferently in the recipient cell compared to the donor cell (e.g.,differentially expressed genes) based on gene expression profiling havetheir expression changed to the level observed for the donor cell.

Phenotype conversion in the recipient cell is maintained stably forextended periods of time. In one embodiment, phenotype conversion isstable and persists for at least about 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, or more. In one embodiment, phenotype conversion is stable for atleast about 1 week, 2 weeks, 3 weeks, 4 weeks, or more. In anotherembodiment, phenotype conversion is stable for at least about 1 month, 2month, 3 months or more. In preferred embodiments, phenotype conversionis stable for the duration of the recipient cell's lifespan or thelifespan of a culture derived from the recipient cell.

Phenotype-converting nucleic acid may comprise two or more nucleic acidshaving different sequences. In some embodiments, the two or more nucleicacids encode different polypeptides. In other embodiments, the nucleicacids are non-coding RNAs or other non-coding nucleic acids. In yetother embodiments, the nucleic acids comprise a mixture of coding andnon-coding nucleic acids. In preferred embodiments, thephenotype-converting nucleic acid comprises the transcriptome,preferably the mRNA transcriptome, from a donor cell. In otherembodiments, the phenotype-converting nucleic consists only of thetranscriptome or mRNA transcriptome from a donor cell. Nucleic acids maybe obtained from a donor cell or may be chemically synthesized or acombination thereof. Methods for chemically synthesizing a nucleic acidare disclosed elsewhere herein and can include in vitro transcription.

An mRNA transcriptome may comprise mRNAs encoding 3 or more, 5 or more,10 or more, 20 or more, 40 or more, 50 or more, 75 or more, 100 or more,200 or more different polypeptides.

The method of the invention may be carried on a cell comprising acellular process. Such a cellular process includes, but is not limitedto, an electrical property such as an action potential, a dendrite, anaxon, a microvilli, a cilia, a stereocilia, a process, an astrocyticprocess, and the like. As demonstrated herein, this methodadvantageously permits the introduction of a desired amount of nucleicacid into one or more local sites, permitting the controlled andlocalized production of protein in physiological amounts, resulting in amultigenic effect in a cell. This method thus allows specificlocalization of exogenously applied nucleic acid, preferably mRNA,without resorting to severing the cellular process from the cell towhich it is attached (Kacharmina, et al., 2000, Proc. Nat'l Acad. Sci.USA, 97:11545-11550). Further, the present method permits the expressionof an mRNA transcriptome of a donor cell, thus resulting in phenotypeconversion in the recipient cell.

The present invention further comprises methods for transfecting a liveslice of tissue or a live animal. Methods for sustaining the cellularprocesses in the cells comprising a live slice of tissue are known inthe art. As a non-limiting example, live slices can be refrigerated andperfused with natural or artificial fluids, such as artificial spinalfluid, artificial central nervous system fluid, and buffers disclosedelsewhere herein. Methods for the manipulation of live slice culturesare described in, for example, Roelandse, et al. (2004, J. Neuroscience,24: 7843-7847); and Chen, et al. (2005, Magn. Reson. Med. 53: 69-75).

Methods for transfecting a live animal, preferably a mammal, areperformed using the methods described herein combined with methods ofanimal and human surgery known in the art. Exemplary surgical procedurescontemplated for use with the methods of the invention include cardiaccatherization, angioplasty, arthroscopy, laparoscopy, tumor resection,surgical placement of a therapeutic implant and the like. Mammalscontemplated in the present invention include, but are not limited to,mice, rabbits, rats, goats, guinea pigs, humans, and the like.

As a non-limiting example, one or more nucleic acids is applied to atissue in a live animal to transfect the tissue in the live animal withone or more nucleic acids. The nucleic acid is introduced to the animalusing methods disclosed elsewhere herein, such as through a microscopeor an optical fiber or endoscopy. The expression of a polypeptidetransfected using the methods of the present invention is monitoredusing methods of detecting protein expression known in the art, such asWestern blots, immunocytochemistry, in situ protein detection, and thelike. Methods for using a laser to manipulate animal tissues are wellknown in the art and are described in, for example, Dang, et al. (2005,Exp Dermatol., 14: 876-882).

The methods disclosed herein comprise introducing phenotype-convertingnucleic acid, preferably RNA and more preferably mRNA, siRNA, miRNA,hnRNA, tRNA, non-coding RNAs and combinations thereof, including but notlimited to total mRNA, to a cell and transfecting the cell at one ormore sites on the cell membrane. Preferably, the phenotype-convertingnucleic acid introduced into a cell is an mRNA transcriptome. The cellis preferably a primary cell culture or in slice culture. The cell canbe transfected at any site. The nucleic acid can be provided to the cellby any method known to the skilled artisan, and is preferably providedby means of a nucleic acid bath comprising a mixture of nucleic acids,disclosed elsewhere herein. A nucleic acid bath is a solution comprisinga nucleic acid of interest in which a cell is bathed. In one embodiment,bath application of the cell comprises surrounding the cell with asolution comprising nucleic acid, thus bathing the entire cell. In oneembodiment, the cell is then irradiated with a laser at one or moresites located anywhere on the cell. In another embodiment, bathapplication comprises bathing a discrete portion or portions of a livecell, for instance, by applying a solution comprising nucleic acid to adiscrete location on the surface of the cell. In one embodiment, thecell is then irradiated one or more times within the discrete locationor locations that was bathed. The discrete location bath is advantageousbecause it creates a greater mRNA concentration gradient, which allowsmRNAs to diffuse more efficiently through the temporary poration holesinto the porated cell. It also requires less mRNAs (e.g., 0.3 μg) thanthe bath application (e.g., 20 μg). In either case, the solution isappropriately buffered and is of the proper pH to maintain thestructural integrity of the cell to be transfected.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a cell includecalcium phosphate precipitation, lipofection, particle bombardment,microinjection, electroporation, phototransfection and the like. Methodsfor producing cells comprising vectors and/or exogenous nucleic acidsare well-known in the art. See, for example, Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL volumes 1-3 (3rd ed., Cold Spring HarborPress, NY 2001).

Biological methods for introducing a polynucleotide of interest into acell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome {e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St, Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −200 C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Phenotype-converting nucleic acid suitable for use in the method of theinvention may be of any size. For instance, a nucleic acid of about 800nucleotides and a nucleic acid of about 3000 nucleotides have beensuccessfully transfected into cells. However, the methods of the presentinvention are not limited to a nucleic acid, preferably an RNA, of thesizes disclosed herein. The present invention comprises transfecting anucleic acid of about 30 bases, even more preferably, about 50 bases,yet more preferably, about 75 bases, even more preferably, about 100bases, yet more preferably, about 75 bases, even more preferably, about100 bases, yet more preferably, about 150 bases, even more preferably,about 200 bases, yet more preferably, about 300 bases, even morepreferably, about 500 bases, yet more preferably, about 750 bases, evenmore preferably, about 1000 bases, yet more preferably, about 1500bases, even more preferably, about 2000 bases, yet more preferably,about 2500 bases, even more preferably, about 3000 bases, in length.Even more preferably, the present invention comprises transfecting,sometimes by phototransfection, a mixture of RNAs encoding differentproteins and of different molecular weights. In preferred embodiments,the phenotype-type converting nucleic acid is an mRNA transcriptomehaving a range of mRNA transcript sizes and having an average mRNAtranscript size from about 0.5 kb to about 5 kb, more preferably, fromabout 1 kb to about 3.5 kb. As a non-limiting example, the mRNAtranscriptome is obtained from cardiomyocyte, wherein the average sizeof the mRNA transcriptome is about. In some embodiments, thetranscriptome is transfected into a recipient cell, such as afibroblast, to induce phenotype conversion of the fibroblast to thetCardiomyocyte phenotype. In other embodiments, the transcriptome istransfected into a recipient cell, such as a fibroblast, to inducephenotype conversion of the fibroblast to the induced pluripotent stem(iPS) cell phenotype.

As another non-limiting example, a nucleic acid expression profile of acell in a desired physiological state (e.g. during differentiation, in adisease state, after treatment with a pharmaceutical, toxin,transcription inhibitor, or other compound) and a nucleic acidexpression profile of a cell in another physiological state (e.g. thesame cell type pre- or post-differentiation, not in a disease state, orbefore treatment with a pharmaceutical, toxin, transcription inhibitoror other compound) can be obtained using techniques for RNA isolationknown in the art and disclosed elsewhere herein. The cDNA clones ofthese RNAs can be generated, reflecting the altered RNA abundances ofthe differing physiological states, or the RNA can be transfected into acell without first reverse transcribing the RNA to cDNA. These RNA canbe mixed according to the same ratios and abundances indicated by thenucleic acid expression profiles of the cells in differing physiologicalstates. These nucleic acid mixtures are then transfected into a cellusing the transfection methods disclosed herein, and those known in theart. The methods of the present invention permit the local transfectionof a cell, and therefore the nucleic acid mixture can be locallytransfected to a specific part of a cell, or the nucleic acid mixturecan be generally transfected into a cell by transfecting any portion ofthe cell. Using the methods of the present invention, and thephysiologically relevant mixtures of nucleic acids described herein,once the mixture of nucleic acids is expressed in a cell, the phenotypeof the physiological state can be replicated in a cell or a cellularprocess, thus allowing the skilled artisan to observe the phenotypetransfer in a cell or cellular process.

Nucleic acid, preferably a transcriptome, may be obtained from any cellof interest in any physiological state, such as, for example, acardiomyocyte. The donor cell may be any type of cell. A donor cell maybe a eukaryotic cell or a prokaryotic cell. When the cell is aeukaryotic cell, the cell is preferably a mammalian cell, including butnot limited to human, non-human primate, mouse, rabbit, rat, goat,guinea pig, horse cell, and the like. A non-mammalian eukaryotic cellincludes a yeast cell, a plant cell, an insect cell, a protozoan celland a fungal cell, including filamentous and non-filamentous fungi. Whenthe cell is a prokaryotic cell the cell is a bacterial cell.Non-limiting examples of cells from which nucleic acid may be obtainedinclude astrocytes, cardiomyocytes, neonatal cardiomyocytes, embryonicstem cells and neurons. RNA from any donor cell of interest can betransfected into any recipient cell in the method of the invention, suchas, for example, a fibroblast. Preferably, donor cells are of the samespecies as the recipient cells. Donor cells may be from the sameindividual as the recipient cell, or from a different individual. Donorcells may originate from the same germinal layer (e.g., ectoderm) as therecipient cell (e.g. both arise from ectoderm germ layer), or from adifferent germinal layer (e.g., one cell arises from ectoderm and theother arises from endoderm germ layer). Donor cells may be the same celltype as the recipient cell but at a different stage of differentiation,exposed to a candidate therapeutic, exposed to a toxin or pathogen,diseased. In yet other embodiments, a donor cell may be a recipientcell. For instance, nucleic acid from a donor cell is transferred into afirst recipient cell. Nucleic acid from the first recipient cell is thensubsequently transferred into a second recipient cell. In one aspect,the first and second recipient cells are in different physiologicalstates. In another aspect, the first and second recipient cells are thesame type of cell. As described elsewhere herein, RNA obtained from acell may be used to transfect cell, or may be used as a template tocreate cDNA. The cDNA may be used in in vitro transcription methods toamplify some or all of the RNA, which is then used in the method of theinvention.

As a non-limiting example, the total RNA from a cardiomyocyte or otherprogenitor cardiomyocyte cell can be isolated from such a cell usingtechniques known in the art and disclosed elsewhere herein, To obtain anmRNA transcriptome, the total RNA can then be processed using variousmethods known in the art for isolating mRNA, such as isolation of mRNAusing complementary poly-dT nucleic acids, which can be conjugated tobeads or a column. The total mRNA obtained is then transfected into arecipient cell using the methods disclosed herein. The recipient cellthen expresses the mixture of mRNA isolated from the cardiomyocyte andreplicates the multigenic effect of the differential gene translationand regulation characteristic of a developing cardiomyocyte. The presentinvention is not limited to cardiomyocytes or their progenitors however,and can be used to determine the transferred multigenic phenotype of anytype of developing or developed cell, provided that the total RNA andmRNA are isolated from the cell.

As non-limiting example, the total RNA from a cell treated with acompound, such as a drug, a peptide, a cytokine, an antibody, a mitogen,a toxin, a transcription inhibitor or other compounds known in the art,can be isolated using the methods disclosed herein and known in the art.The mRNA from that cell can then be transfected into another cell typeusing the methods disclosed herein, thus transferring the multigenicphenotype of the cell treated with a compound to another cell, thusenabling the rapid and specific determination of that compound onanother cell type.

In another non-limiting embodiment of the present invention, the totalRNA from a diseased cell, such as a tumor cell, a cell harboring anintracellular pathogen, a cell from a patient with an autoimmunedisease, and the like, can be isolated from the diseased cell. The mRNAtranscriptome from that cell can be isolated from the total RNA using,for example, poly-dT isolation techniques. The mRNA from the diseasedcell is transfected into another cell using the methods of the presentinvention, thus transferring the multigenic phenotype of the diseasedcell to another cell, providing a more accurate picture of the roleinteracting nucleic acids and their encoded proteins have in thephenotype of a cell.

As another non-limiting embodiment of the invention, the method of theinvention can be practiced in order to prepare cells for testingtherapeutics. Candidate therapeutics are typically tested on a number ofdifferent cell types, prior to assessment in animals or humans. Thesedifferent cells often are cell lines that have a multiplicity ofsignaling pathways. The multiplicity of pathways may overlap andcompensate for drug function and testing with regard to efficacy and/orside effects, thereby making assessment of the candidate drug effectsless robust. According, it is contemplated that mRNA for one or morespecified second messenger system pathways can be transfected intoprimary cells or cell lines of interest in order to create cells havingenriched presence and/or activity of one or more pathways, thus thesepathways will dominate over endogenous pathways. The mRNA are thereforea heterogeneous collection of mRNAs that encode the various componentsfor the one or more second messenger system pathways. Enriched presenceand/or activity of one or more pathways is relative to a cell that hasnot had mRNA for one or more specified second messenger system pathwaystransfected into it. Candidate therapeutics can then be assessed forefficacy and/or side effects on the dominant pathways present in thecells with enriched expression of one or more specified second messengersystem pathways. Non-limiting examples of second messenger systemsinclude: the cAMP system; the phosphoinositol system; the arachidonicacid system; the cGMP system; and the tyrosine kinase system. It isexpected that using such defined cell types permits improved assessmentof the effect of a candidate on particular pathways. In one embodiment,modulation of endogenous pathways by decreasing expression of particularpathways is also contemplated. Modulation can be achieved by introducingsiRNAs corresponding to mRNAs encoding particular proteins in a pathwayinto the cell to inhibit particular pathways. Such modulation can beperformed simultaneously with the introduction of the mRNAs for the oneor more specified second messenger system pathways, or can be done inone or more separate steps. In one embodiment, an embryonic fibroblastis used as the recipient cell. In one embodiment, the donor cells fromwhich total RNA is obtained are cardiomyocytes and the recipient celltype is an embryonic fibroblast. In a preferred aspect, mRNA isextracted from the cardiomyocyte total RNA and is transfected into theembryonic fibroblast.

In another non-limiting embodiment, the method of the invention can beused to generate a knock out (KO) of one or more specific genes in acell. The field of functional genomics has relied upon the generation ofKO mice to elucidate the function of particular genes. The utility of KOmice has been enhanced by flanking a gene with FLOX-sites, which arerecognized by CRE-recombinase. CRE-recombinase binds to FLOX-sites andremoves the intervening sequence containing the gene, thereby knockingout that gene. Cell-type specific KO has been achieved by drivingCRE-recombinase expression in particular cell types using cell-typespecific promoters. Inducible promoters, such as TET-on or the ecdysonesystem, have been used to control the time of induction ofCRE-recombinase expression; expression is induced upon exogenousaddition of the cognate inducer. Advantageously, the method of theinvention can be used to knock out a gene in a particular cell at aparticular time without the use of inducible promoters and exogenousinducers. In one embodiment, mRNA encoding CRE-recombinase, or theprotein itself, is transfected into cells having chromosomal materialengineered genetically to contain FLOX sites flanking one or more genesof interest. The transfection can be done with a single engineered cellor with a population of the engineered cells. The method can also bepracticed with live tissue samples or with a live animal. The method isnot limited to the use of CRE-recombinase and FLOX sites. It can bepracticed using any comparable system of specific sequence excision,such as zinc-finger nuclease technology and the FLP recombinase and FRTsystem. The method can also be used for targeted integration of a gene.

The present invention can further comprise the use of a nucleic acidfrom a cell or a population of cells of homogeneous or heterogeneoustypes. The present invention can further comprise the use of a nucleicacid, preferably mRNA, defined by the expression profile of a cell asdetermined using methods well known in the art, including, but notlimited to, a gene array profile, total RNA, total mRNA, and the like.An expression profile is used to determine the relative abundances ofmRNA in a cell. The expression profile is then used as a template todetermine the relative abundances of mRNA in the physiological state ofthe cell from which the expression profile was made. A population ofmRNA with the same relative abundance as in the cell for whichexpression has been profiled is produced using the methods disclosedelsewhere herein, including mRNA isolation, in vitro transcription orchemical synthesis. The resultant population of mRNA is then transfectedinto the cell using the methods described elsewhere herein, therebytransferring the phenotype of the cell from which the expression profilewas made to another cell, tissue or animal.

In another embodiment, a population of mRNA reflecting the relativeabundance of a cell in a particular physiological state furthercomprises mRNA encoding one or more polypeptides that facilitatephenotype conversion. For instance, the mRNA obtained from a neuronalcell may be supplemented with mRNA encoding proteins that stimulateexocytosis and is then transfected into a non-neuronal recipient cell.

The present invention may further comprise the sequential transfectionof a cell, Sequential transfection is used herein to refer to a processin which a cell is transfected at a first time point, and thentransfected at a second or subsequent time point. As an example, a cellcan be transfected on day 1, the result of which is that one or morenucleic acids are introduced into the cell. These nucleic acids can beexpressed by the cellular translation complexes or remain silent, or canbe inhibited using an inhibitory nucleic acid as disclosed elsewhereherein. On day 2, the same cell can be transfected again, transfectingone or more of the same or dissimilar nucleic acids to the same cell.The present invention is not limited to transfection separated by a dayhowever. Sequential transfection can occur with minutes, hours, days,weeks or months between a first time point and a second time point,provided the transfection occurs to the same cell. Thus, the sequentialtransfection methods of the present invention are limited only by thelifespan of the cell. Another non-limiting example of sequentialtransfections comprises a first transfection on Day 1, a secondtransfection 48 hours later (Day 3) and a third transfection 7 daysafter the first transfection. The conditions of sequential transfectionmay be the same or different. The means of transfection may be changedand/or the number of sites transfected in a transfection step may bedifferent among multiple transfections. For instance, the second andsubsequent transfections using transfection may be performed using areduced laser power compared to the laser power used in the firsttransfection.

The sequential transfection methods of the present application areuseful for, among other things, analyzing temporal gene expression in acell, analyzing the multigenic effects of a protracted developmentalprocess, and determining the relationship of genotype to phenotype overthe course of the viable life span of a cell. Sequential transfectionusing the same nucleic acids also increases the robustness of expressionof the phototransfected nucleic acids. As shown herein, three sequentialtransfections of cardiomyocyte transcriptome into a fibroblast yields adurable phenotype conversion in a high percentage of fibroblast cells.

The embodiments of the inventions disclosed herein are not limited tomRNA. The present invention can further comprise reverse transcribingmRNA into cDNA, then transfecting the cDNA into a cell

The present invention is not limited to the use of RNA and mRNA. Amixture of DNA and RNA can be used in the methods of the presentinvention to determine the effects of transient (RNA) as well asprolonged (DNA integration into the genome) gene expression in a cell.

When a mixture of nucleic acids, such as a mixture of RNAs istransfected into a cell, subpopulations of that mixture can betransfected into a cell to determine the core set of RNAs responsiblefor a given phenotype. As a non-limiting example, when the total RNA isisolated from a cell in a certain physiological state and mRNA isisolated from that population of total RNA, specific subpopulations ofthe isolated mRNA can be transfected into a cell to establish the coremRNAs responsible for that phenotype. The present embodiment can also beperformed with cDNA produced from mRNA. Specific populations of mRNA canbe identified using sequence homology data or other characteristicfeatures known in the art and available from various databases, such asGenBank® (United States Department of Health and Human Services,Bethesda Md.).

Alternatively, the mRNA from a cell can be isolated and transfected intoa cell using the methods of the present invention, and an siRNA,microRNA, antisense nucleic acid or ribozyme (collectively referred toas an inhibitory nucleic acid) can be transfected along with the mRNA,resulting in silencing and/or inhibition of an mRNA. Silencing an mRNApermits one of skill in the art to identify, for instance, the coremRNA(s) responsible for a multigenic phenotype. In addition, the presentinvention allows the replication of a phenotype in another cell withoutthe step of determining the nucleic acid expression profile of a cell ina physiological state. The nucleic acid, preferably RNA, from a cell ina specific physiological state, such as a certain differential ordisease state, can be isolated. Preferably, an mRNA transcriptome isthen isolated. Using the methods of the present invention, the RNA, or acDNA of the RNA, can be transfected into a cell in order to analyze thephenotype in the transfected cell once the nucleic acid has beenexpressed. The nucleic acid, preferably RNA, can be the total RNA from acell, or a subpopulation of the RNA, such as the mRNA transcriptome.

To assess the effect of expression of the transfected nucleic acids,cells transfected in accordance with the method of the invention can beexamined using methods known in the art. Assessments may be made, forexample, of phenotypic changes, mRNA expression, protein expression andfunctional assays. Examples of such analyses include, but are notlimited to, cell morphology, presence and absence of immunologicalmarkers, RT-PCR, expression profiling, mRNA abundance measurements,immunocytochemistry analysis (ICC) for specific proteins, cellviability, and cell-specific activities, such as cell division-mitosisand electrophysiology.

Optionally, the present method further comprises inhibitingtranscription in the transfected cell, thus preventing competitionbetween expression of endogenous and exogenous mRNAs and the proteinsencoded thereby. Transcription can be inhibited by addition of exogenousagents, such as an inhibitory nucleic acid or compounds that inhibittranscription, such as 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole(DRB), a protease, or SP100030 (Huang et al., 2001, Br. J. Pharmacol.,134: 1029-1036). Other agents useful for inhibiting transcription in arecipient cell include, but are not limited to, α-amanitin, trichostatinA (TSA; a histone deacetylase inhibitor), tubulin depolymerizer andactin depolymerizer. Preferably, a recipient cell is contacted with oneor more transcription inhibition agents prior to transfection.Preferably, the cell is contacted between about 30 minutes and about 80hours, preferably between about 30 minutes and about 60 hours and morepreferably, between about 6 hours to about 48 hours, prior totransfection.

The present method can also be used for the specific and localtransfection of an inhibitory nucleic acid, such as an siRNA, antisensenucleic acid or a microRNA (miRNA), using the methods of the presentinvention. Using the invention disclosed herein, the skilled artisan canspecifically inhibit a cellular nuclear acid, especially those incellular processes. Further, as disclosed elsewhere herein, aninhibitory nucleic acid can be used to identify the core nucleic acid(s)involved in a multigenic phenotype.

The phenotype-converting nucleic acids useful in the methods of thepresent invention may comprise a variety of nucleic acids, includingvarious species of RNA (mRNA, siRNA, miRNA, hnRNA, tRNA, total RNA,combinations thereof and the like) as well as DNA. Methods for isolatingRNA from a cell, synthesizing a short polynucleotide, constructing avector comprising a DNA insert, and other methods of obtaining a nucleicacid to phototransfect into a cell are well known in the art andinclude, for example, RNA isolation, cDNA synthesis, in vitrotranscription, and the like.

The nucleic acid compositions of this invention, whether RNA, cDNA,genomic DNA, or a hybrid of the various combinations, may be isolatedfrom natural sources or may be synthesized in vitro. Techniques fornucleic acid manipulation are described generally in Sambrook et al.(2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York), and in Ausubel et al. (1997, Current Protocols inMolecular Biology, John Wiley & Sons, New York)., incorporated herein byreference. Nucleic acids suitable for use in the present method alsoinclude nucleic acid analogs. Examples of such analogs include, but arenot limited to, phosphorothioate, phosphotriester, methyl phosphonate,short chain alkyl or cycloalkyl intersugar linkages, or short chainheteroatomic or heterocyclic intersugar (“backbone”) linkages. Inaddition, nucleic acids having morpholino backbone structures (U.S. Pat.No. 5,034,506) or polyamide backbone structures (Nielsen et al., 1991,Science 254: 1497) may also be used.

The methods of the present invention can comprise the use of a varietyof nucleic acids, including DNA, RNA, a cDNA reverse transcribed from anmRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA,an RNA transcribed from the amplified DNA, and the like. The presentinvention further comprises using single-stranded and double-strandedRNA and DNA molecules. Any coding sequence of interest can be used inthe methods of introducing and translating a nucleic acid in a cell orin a cellular process, such as a dendrite. One of skill in the art willunderstand, when armed with the present disclosure, that a multitude ofproperties of a cellular process, and by association, of the attachedcell, can be affected by the methods of the present invention.

In one embodiment of the present invention, the nucleic acid transfectedinto a cell is all or a portion of the total mRNA isolated from abiological sample. The term “biological sample,” as used herein, refersto a sample obtained from an organism or from components (e.g., organs,tissues or cells) of an organism. The sample may be of any biologicaltissue or fluid. The nucleic acid (either genomic DNA or mRNA) may beisolated from the sample according to any of a number of methods wellknown to those of skill in the art.

Methods of isolating total mRNA are well known to those of skill in theart. For example, methods of isolation and purification of nucleic acidsare described in detail in Chapter 3 of Laboratory Techniques inBiochemistry and Molecular Biology: Hybridization With Nucleic AcidProbes, Part 1. Theory and Nucleic Acid Preparation, P. Tijssen, ed,Elsevier, N.Y. (1993) and Chapter 3 of Laboratory Techniques inBiochemistry and Molecular Biology Hybridization With Nucleic AcidProbes, Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed.Elsevier, N.Y. (1993)).

Preferably, the total nucleic acid is isolated from a given sampleusing, for example, an acid guanidinium-phenol-chloroform extractionmethod and polyA⁺ mRNA is isolated by oligo dT column chromatography orby using (dT)_(n) magnetic beads. Commercially available products, suchas TRIZOL and MICRO-FASTTRACK (Invitrogen™, Carlsbad, Calif.), areuseful in extracting nucleic acid from a biological sample.

The mRNA can be locally transfected directly into a cell or a cellularprocess, or the sample mRNA can be reverse transcribed with a reversetranscriptase and a promoter comprising an oligo dT and a sequenceencoding the phage T7 promoter to provide single stranded DNA template.The second DNA strand is polymerized using a DNA polymerase. Aftersynthesis of double-stranded cDNA, T7 RNA polymerase is added and RNA istranscribed from the cDNA template. Successive rounds of transcriptionfrom each single cDNA template results in amplified RNA. Methods of invitro polymerization are well known to those of skill in the art (see,e.g., Sambrook, supra.; Van Gelder, et al., 1990, Proc. Natl. Acad. Sci.USA, 87: 1663-1667), Moreover, Eberwine et al. (1992, Proc. Natl. Acad,Sci. USA, 89: 3010-3014) provide a protocol using two rounds ofamplification via in vitro transcription to achieve greater than 10⁶fold amplification of the original starting material.

The present invention further comprises the use of in vitrotranscription for transfection into a cell or cellular process. In vitrotranscription comprises the production of dsRNA by transcribing anucleic acid (DNA) segment in both directions. For example, theHiScribe™ RNAi transcription kit (New England Biolabs, Ipswich, Mass.)provides a vector and a method for producing a dsRNA for a nucleic acidsegment that is cloned into the vector at a position flanked on eitherside by a T7 promoter. Separate templates are generated for T7transcription of the two complementary strands for the dsRNA. Thetemplates are transcribed in vitro by addition of T7 RNA polymerase anddsRNA is produced. Similar methods using PCR and/or other RNApolymerases (e.g., T3 or SP6 polymerase) can also be used and are knownin the art.

The present invention further comprises the use of chemicallysynthesized nucleic acids for use in transfection. Oligonucleotides foruse as probes can be chemically synthesized according to the solid phasephosphoramidite triester method first described by Beaucage, (1981,Tetrahedron Letts., 22:1859-1862) using an automated synthesizer, asdescribed in Needham-VanDevanter, et al. (1984, Nucleic Acids Res.,12:6159-6168). Purification of oligonucleotides is by either nativeacrylamide gel electrophoresis or by anion-exchange HPLC as described inPearson (1983, J. Chrom., 255:137-149). The sequence of the syntheticoligonucleotides can be verified using the chemical degradation methodof Maxam (1980, in Grossman and Moldave, eds., Methods in Enzymology,Academic Press, New York, 65:499-560).

The present invention can further comprise the use of DNA in a processto locally transfect a cell or a cellular process via transfection. TheDNA can be contained in a vector.

The invention includes an isolated DNA encoding a protein operablylinked to a nucleic acid comprising a promoter/regulatory sequence suchthat the nucleic acid is preferably capable of directing expression ofthe protein encoded by the nucleic acid. Thus, the invention encompassesexpression vectors and methods for the introduction of exogenous DNAinto cells with concomitant expression of the exogenous DNA in the cellssuch as those described, for example, in Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York), and in Ausubel et al. (1997, Current Protocols in MolecularBiology, John Wiley & Sons, New York).

Expression of a protein in a cell or a cellular process transfected asdisclosed herein may be accomplished by generating a plasmid or othertype of vector comprising the desired nucleic acid operably linked to apromoter/regulatory sequence which serves to drive expression of theprotein, with or without a tag, in cells in which the vector isintroduced. Many promoter/regulatory sequences useful for drivingconstitutive expression of a gene are available in the art and include,but are not limited to, for example, the cytomegalovirus immediate earlypromoter enhancer sequence, the SV40 early promoter, as well as the Roussarcoma virus promoter, and the like. Moreover, inducible and tissuespecific expression of the nucleic acid encoding a protein can beaccomplished by placing the nucleic acid encoding a protein under thecontrol of an inducible or tissue specific promoter/regulatory sequence.Examples of tissue specific or inducible promoter/regulatory sequenceswhich are useful for his purpose include, but are not limited to theMMTV LTR inducible promoter, and the SV40 late enhancer/promoter. Inaddition, promoters which are well known in the art which are induced inresponse to inducing agents such as metals, glucocorticoids, and thelike, are also contemplated in the invention. Thus, it will beappreciated that the invention includes the use of anypromoter/regulatory sequence, which is either known or unknown, andwhich is capable of driving expression of the desired protein operablylinked thereto.

Selection of any particular plasmid vector or other DNA vector is not alimiting factor in this invention and a wide plethora of vectors arewell-known in the art. Further, it is well within the skill of theartisan to choose particular promoter/regulatory sequences and operablylink those promoter/regulatory sequences to a DNA sequence encoding adesired polypeptide. Such technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inAusubel et al. (1997, Current Protocols in Molecular Biology, John Wiley& Sons, New York).

The nucleic acids encoding a protein can be cloned into various plasmidvectors. However, the present invention should not be construed to belimited to plasmids or to any particular vector. Instead, the presentinvention should be construed to encompass a wide plethora of vectorswhich are readily available and/or well-known in the art.

The present invention further comprises locally transfecting aninhibitory nucleic acid, such as an antisense nucleic acid, an siRNA oran miRNA into a cell. An siRNA polynucleotide is an RNA nucleic acidmolecule that interferes with RNA activity that is generally consideredto occur via a post-transcriptional gene silencing mechanism. An siRNApolynucleotide preferably comprises a double-stranded RNA (dsRNA) but isnot intended to be so limited and may comprise a single-stranded RNA(see, e.g., Martinez et al., 2002, Cell 110:563-74). The siRNApolynucleotide included in the invention may comprise other naturallyoccurring, recombinant, or synthetic single-stranded or double-strandedpolymers of nucleotides (ribonucleotides or deoxyribonucleotides or acombination of both) and/or nucleotide analogues as provided herein(e.g., an oligonucleotide or polynucleotide or the like, typically in 5′to 3′ phosphodiester linkage). Accordingly it will be appreciated thatcertain exemplary sequences disclosed herein as DNA sequences capable ofdirecting the transcription of the siRNA polynucleotides are alsointended to describe the corresponding RNA sequences and theircomplements, given the well-established principles of complementarynucleotide base-pairing.

An siRNA may be transcribed using as a template a DNA (genomic, cDNA, orsynthetic) that contains a promoter for an RNA polymerase promoter. Forexample, the promoter can be the U6 promoter or the H1 RNA polymeraseIII promoter. Alternatively, the siRNA may be a synthetically derivedRNA molecule. In certain embodiments, the siRNA polynucleotide may haveblunt ends. In certain other embodiments, at least one strand of thesiRNA polynucleotide has at least one, and preferably two nucleotidesthat “overhang” (i.e., that do not base pair with a complementary basein the opposing strand) at the 3′ end of either strand of the siRNApolynucleotide. In a preferred embodiment of the invention, each strandof the siRNA polynucleotide duplex has a two-nucleotide overhang at the3′ end. The two-nucleotide overhang is preferably a thymidinedinucleotide (TT) but may also comprise other bases, for example, a TCdinucleotide or a TG dinucleotide, or any other dinucleotide. Theoverhang dinucleotide may also be complementary to the two nucleotidesat the 5′ end of the sequence of the polynucleotide that is targeted forinterference. For a discussion of 3′ ends of siRNA polynucleotides see,e.g., WO 01/75164.

Preferred siRNA polynucleotides comprise double-stranded polynucleotidesof about 18-30 nucleotide base pairs, preferably about 18, about 19,about 20, about 21, about 22, about 23, about 24, about 25, about 26, orabout 27 base pairs, and in other preferred embodiments about 19, about20, about 21, about 22 or about 23 base pairs, or about 27 base pairs.The siRNA polynucleotide useful in the present invention may alsocomprise a polynucleotide sequence that exhibits variability bydiffering (e.g., by nucleotide substitution, including transition ortransversion) at one, two, three or four nucleotides from a particularsequence. These differences can occur at any of the nucleotide positionsof a particular siRNA polynucleotide sequence, depending on the lengthof the molecule, whether situated in a sense or in an antisense strandof the double-stranded polynucleotide. The nucleotide difference may befound on one strand of a double-stranded polynucleotide, where thecomplementary nucleotide with which the substitute nucleotide wouldtypically form hydrogen bond base pairing, may not necessarily becorrespondingly substituted. In preferred embodiments, the siRNApolynucleotides are homogeneous with respect to a specific nucleotidesequence.

Polynucleotides that comprise the siRNA polynucleotides may in certainembodiments be derived from a single-stranded polynucleotide thatcomprises a single-stranded oligonucleotide fragment (e.g., of about18-30 nucleotides) and its reverse complement, typically separated by aspacer sequence. According to certain such embodiments, cleavage of thespacer provides the single-stranded oligonucleotide fragment and itsreverse complement, such that they may anneal to form, optionally withadditional processing steps that may result in addition or removal ofone, two, three or more nucleotides from the 3′ end and/or the 5′ end ofeither or both strands, the double-stranded siRNA polynucleotide of thepresent invention. In certain embodiments the spacer is of a length thatpermits the fragment and its reverse complement to anneal and form adouble-stranded structure (e.g., like a hairpin polynucleotide) prior tocleavage of the spacer, and optionally, subsequent processing steps thatmay result in addition or removal of one, two, three, four, or morenucleotides from the 3′ end and/or the 5′ end of either or both strands.A spacer sequence may therefore be any polynucleotide sequence asprovided herein that is situated between two complementarypolynucleotide sequence regions which, when annealed into adouble-stranded nucleic acid, result in an siRNA polynucleotide.

The present method further comprises methods for introducing a nucleicacid into a cell. The method comprises transfecting a cell in thepresence of a nucleic acid, preferably RNA and/or DNA, where the nucleicacid is in a fluid medium permitting the transfer of the nucleic acidfrom one side of the cell membrane to the other side of the cellmembrane through the cell membrane. The fluid medium can comprise anymedium having the buffering capacity and pH to support the viability ofa cell and the stability of a nucleic acid molecule. Contemplated mediainclude, but are not limited to, phosphate buffered saline, Tris,Tris-EDTA (TE) cell culture media, other aqueous mediums and buffers,and the like.

The number of nucleic acid molecules that enter the cell is influencedby the nucleic acid concentration in the nucleic acid bath, the size ofthe nucleic acid molecule, and, with photo transfection, the laserintensity, e.g., the length of each laser pulse and the number of laserpulses delivered. Based on the teachings herein, the skilled artisan canreadily adjust the parameters of the transfection process to control theapproximate number of nucleic molecules that enter the cell.

In one embodiment, a cell is surrounded by an nucleic acid bathcomprising a nucleic acid molecule, preferably an RNA molecule, at about1 to about 150 μg/ml, more preferably about 10 to about 100 μg/ml, andmore preferably still at about 10 to about 50 μg/ml in the bath.

In another embodiment, a cell is bathed in discrete locations on thecell surface with a solution comprising a nucleic acid molecule. Forinstance, using a patch pipette, micropipette or other applicator, asolution comprising nucleic acid is applied to a discrete location onthe surface of a cell. The solution may be applied to more than onelocation on a cell. If phototransfection is employed, the cell is thenirradiated using a laser at one or more sites within a discretelocation, Nucleic acid in the solution is present at about 1 nanogramper microliter (ng/μl) to about 2 microgram/microliter (μg/μl),preferably about 50 ng/μl to about 1 μg/μl, and more preferably about100 ng/μl to about 500 ng/μl.

The present invention further comprises the use of other methods forintroducing a nucleic acid to a cell, tissue or animal via transfection.Methods included in the present invention include, for example,perfusion, picospritzing, microinjection and the like. Methods forperfusion include, but are not limited to, using a pump to move a fluidmedium comprising a nucleic acid, preferably RNA, even more preferablymRNA, to a cell, tissue or animal. The fluid medium used in theperfusion methods of the present invention can included those disclosedelsewhere herein, such as buffered solutions that support and maintainthe stability of a nucleic acid and a cell, tissue or animal. In oneembodiment of the present invention, the fluid medium can include amedium, such as Basal Media Eagle (BME), BGJb Medium, Brinster's BMOC-3Medium, CMRL Medium, CO₂-Independent Medium, Dulbecco's Modified EagleMedia (D-MEM), F-10 Nutrient Mixtures, F-12 Nutrient Mixtures, GlasgowMinimum Essential Media, Grace's Insect Cell Culture Media, ImprovedMEM, IPL-41 Insect Media, Iscove's Modified Dulbecco's Media,Leibovitz's L-15 Media, McCoy's 5A Media (modified), MCDB 131 Medium,Media 199, Medium NCTC-109, Minimum Essential Media (MEM), ModifiedEagle Medium (MEM), Opti-MEMO Reduced Serum Media, RPMI Media 1640,Schneider's Drosophila Medium, Waymouth's MB 752/1 Media, Williams MediaE, artificial spinal fluid (aCSF), Ringer's solution and the like. Thepresent invention can further comprise the use of buffered saltsolutions, including, but not limited to, Dulbecco's Phosphate-BufferedSaline (D-PBS), Earle's Balanced Salt Solution, Hanks' Balanced SaltSolution, Phosphate-Buffered Saline (PBS), and the like.

The present invention further comprises using picospritzing inconjunction with phototransfection to introduce a nucleic acid to acell, organ or tissue. Picospritzing comprises the use of electricalpulses with a pressure device to deliver a compound, such as a nucleicacid, to a cell, tissue or animal. Method for picospritzing are known inthe art and are described in, for example, Herberholz, et al., 2002, J.Neuroscience, 22: 9078-9085). Picospritzing apparatuses are availablefrom, for example, World Precision Instruments (Sarasota, Fla.).

In another embodiment, transfection of cells with nucleic acids encodingtwo or more different polypeptides is effected by microinjection.

When phototransfection is employed, the methods comprise irradiating acell with a laser to phototransfect and locally transfect the cell. Whenthe laser contacts the cell membrane, or cell wall in the case of plantcells, fungal cells, and other cells comprising a cell wall, the plasmamembrane or cell wall is perforated, permitting the diffusion of foreignmolecule, such as RNA and/or DNA, to enter the cell. The fluidity ofmammalian cell membranes facilitates subsequent closure of theperforation. Lasers compatible with the present invention include, butare not limited to, continuous-wave argon-ion lasers operating at 488 nm(Schneckenburger, et al., 2002, J. Biomed. Opt., 7: 410-416; Palumbo etal., 1996, J. Photochem, Photobiol, B-Biol., 36: 41-46), pulsed andfrequency upconverted Nd:YAG lasers operating at 355 nm (Shirahata, etal., 2001, J. invest. Med., 49: 184-190), 532 nm (Soughayer, et al.,2000, Anal. Chem., 72: 1342-1347), and 1064 nm (Mohanty, et al., 2003,Biotechnol. Lett. 25: 895-899), and femtosecond titanium-sapphire lasers(Tirlapur, et al., 2002, Plant J. 31: 365-374; Tirlapur, et al., 2002,Nature 418: 290-291; Zeira, et al., 2003, Mol. Therapy 8: 342-350).Preferably, a titanium-sapphire laser at 405 nm (PicoQuant GmbH, BerlinGermany) is used to phototransfect a cell. However, the presentinvention is not limited to the a titanium-sapphire laser, but includesany laser with the capacity of delivering a localized focal volume ofabout 10⁻¹⁹ m³.

Control of the incident laser beam is achieved by using variousapparatuses to control the focus and power of the laser, as well as toaim the laser. Focusing the laser is achieved by passing the incidentlaser through a lens, such as a microscope lens, placed between thelaser and the cell. The power of the laser in controlled by modulatingthe voltage and current going to the laser and through the use ofneutral density filters or pockels cells. Exposure of the cells to thelaser is controlled through a shutter, such as a single lens reflex(SLR) camera shutter and/or with electronically controlled pockelscells.

Aiming the laser is accomplished through a microscope lens and withdielectric and steering mirrors and AOD (acoustic optical deflector)between the laser source and a cell. A microscope useful in the practiceof the present invention includes, but is not limited to, a confocalmicroscope, a multiphoton excitation fluorescence microscope, a lightmicroscope, and the like. The present method further comprises aimingthe laser using an optical fiber to transmit the laser to a distant ordifficult-to-access area. As a non-limiting example, an optical fiber isused to phototransfect intestinal, neural or cardiothoracic cells in alive animal. Further, the present invention comprises phototransfectinga cell or a population of cells using multiple optical fibers in ananimal. Optical fibers are well known in the art and are described in,for example, U.S. Pat. Nos. 3,711,262 6,973,245.

A laser beam with less than a milliwatt of power for tens ofmilliseconds is sufficient to porate a cell (Paterson, et al., 2005,Optics Express, 13: 595-600). Preferably, the laser has a power densityof about 1200 MWm⁻² and a total power of about 30-55 mW at the backaperture of the lens. Further, in order to provide maximum surface areafor transfection, the laser beam should be highly circular (dx=dy) withbeam diameter of about 2 mm.

The starting power output of the laser is attenuated through the use ofvarious filters, such as a neutral density (ND) filter to reduce thepower to the milliwatt range required for phototransfection with noattendant pathological effects on the target cell. The beam can beexpanded through the use of a telescope where f=100 mm, and directedinto a microscope, such as a light microscope or an oil-immersionmicroscope with a ×100 objective (N.A.=1.25). An SLR shutter between thelaser source and the microscope permits control of the exposure time. Anexposure time of about 40 ms is sufficient to porate a cell withoutattendant damage, but this parameter can be altered to increase ordecrease exposure time.

Target cells in a nucleic acid bath are positioned and focused on bymanipulating the stage of the microscope and/or using dielectric andsteering mirrors and AOD, so the beam is focused on the cell membraneand not towards the nucleus of the cell. When porating a cellularprocess, such as a dendrite, the beam is focused directly on thecellular process.

An exemplary phototransfection protocol comprises at least two andpreferably three sequential phototransfection steps of a recipient cellusing the transcriptome, preferably the mRNA transcriptome, from a donorcell. The mRNA transcriptome comprises a range of mRNA sizes and has anaverage transcript size between about 1 to 3.5 kb. The firstphototransfection step is at about 35 mW using a titanium-sapphire laserand subsequent phototransfections steps are at a lower power, such as 30mW or less. Each phototransfection step involves laser irradiating therecipient cell at numerous, random sites. The number of sites per stepis determined by consideration of the strength of the laser, thediameter of the pores that result in the irradiated site, the averagesize of the transcripts in the mRNA transcriptome and modeling transportof individual transcripts through the pore using Brownian dynamics.After the first phototransfection step, the recipient cell may betransferred to a growth medium specific for the donor cell.

In some embodiments, the cells are transfected with a nucleic acidcomprising a marker that indicates a successful transfection. Suchmarkers are known in the art and include, for example, antibioticresistance and fluorescent proteins. Successful transfection can betracked by the addition of a detectable molecule to the nucleic acidsolution, Such molecules are well known in the art. Preferably, themolecule is non-toxic to the recipient cell, Non-limiting examplesinclude Lucifer yellow and carboxyfluorescein diacetate succinimidylester. Expression of the locally transfected nucleic acid is analyzedaccording to the presence and activity of a marker or the phenotype ofthe cell.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1 Transdifferentiation of Fibroblast to tCardiomyocyte

Cardiomyocyte-like cells (“tCardiomyocytes”) were created from mouseprimary embryonic fibroblasts using the following materials and methods.

Cell Culture and Poly-A+ RNA Transfection

Primary mouse embryonic fibroblast (PMEF-NL; Millipore) culture wasincubated in DMEM supplemented with 10% FBS at 37° C. with 5% CO2. WTadult mouse (strain C57BL/6, 7-9 wk old) ventricular myocytes wereisolated from hearts mounted on a Langendorf apparatus and perfused withCa2+-free Tyrode's solution with collagenase B and D plus protease. Theventricle was dissected, and sections of ventricle tissue were gentlytriturated to dissociate individual myocytes. Mouse cortical astrocyteswere isolated from mouse embryos and cultured in DMEM supplemented with10% PBS, AVM poly-A+ RNA was isolated using TRIzol and theMicro-FastTrack 2.0 Kit (Invitrogen), following the manufacturer'sprotocol. There were two transfection protocols. First, celltranscription was inhibited by adding 80 μM5,6-dichloro-1-β-D-ribofuranosylbenzimidazole for 30 min beforetransfection. Transcription inhibitor-treated cells were transfectedwith 2 μg of poly-A+ RNA per 35-mm culture dish using the TransMessengerTransfection Reagent Kit (Qiagen) following the manufacturer's protocol.Second, cells were incubated with 80 μM5,6-dichloro-1-β-D-ribofuranosylbenzimidazole for 6 h, after which cellsthat had detached from the bottom were collected by centrifugation. Thecollected cells were transfected with 4 μg of poly-A+ RNA usingLipofectamine 2000 (Invitrogen). Transfected cells were retransfected at2-7 d after the first transfection using the TransMessenger TransfectionReagent Kit. Primary mouse embryonic fibroblast poly-A+ RNA was used asa control transfection. The media was changed every 2-3 d, and growthwas observed under a light microscope. Live cell images were obtainedusing the Zeiss LSM510 or LSM 710 Microscope System. Cell length andwidth were measured by drawing straight lines encompassing nuclei onlive cell images using MetaMorph software (Molecular Devices).

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde for 5 min and permeabalized incold methanol for 10 min at −20° C. The fixed cells were incubated inblocking solution (10% goat serum in PBS) for 30 min at room temperatureand then incubated overnight in primary antibodies (1:500 dilution in 1%goat serum in PBS) at 4° C. Alexa Fluor 488—or Alexa Fluor568—conjugated secondary antibodies (1:500 dilution in 1% goat serum inPBS) were used to label primary antibodies. Immunostained cells weremounted on glass slides using DAPI-containing mounting medium. Imageswere captured with the Zeiss LSM 510 or LSM 710 microscope system.

Single Cell Microarray

Poly-A+ RNAs of single cells were isolated and amplified following thestandard single-cell harvesting and aRNA amplification method (1990, VanGelder et al., Proc Natl Acad Sci USA 87:1663-1667). Amplifiedsingle-cell aRNAs (three or four rounds of amplification) were used toprobe the Affymetrix GeneChip Mouse Genome 430 2.0 array. Data wereanalyzed using the R/Bioconductor package and Gene-Spring GX version 11(Agilent) (2003, Irizarry et al., Nucleic Acids Res 31:e15; 2004,Gentleman et al., Genome Biol 5:R80). The second-highest intensityvalues were extracted from the probe sets, and informative probe setswere selected based on their ability to distinguish AVMs fromfibroblasts. Individual t tests were performed for each probe set,contrasting AVM expression (n=4) and fibroblast expression (n=3), andthe 3,257 probe sets with expression differing significantly between thetwo cell types (P<0.05) were retained. Hierarchical clustering wasperformed on these 3,257 probe sets across all cell types (four AVMs,three fibroblasts, five cardio-TIPeR cells, and three fibro-TIPeRcells), using Euclidean distance and the complete linkage method.Bootstrap values were calculated using the R pvclust package; unbiased Pvalues for 1,000 times resampling support of the tree were reported.Heat maps were produced using the heat map.2 routine in the R gplotspackage, focusing on gene subsets for which successfully convertedtCardiomyocytes had significantly (P<0.1) different expression thanuntreated fibroblasts by the t test on each probe set. These lists werefiltered to exclude probe sets with very similar expression (less than atwofold difference) between AVMs and fibroblasts, as well as probe setsfor genes that appeared to be induced by the treatment effect [probesets showing a significant (P<0.05) expression difference betweenfibroblasts and the fibroblast treatment controls]. Gene Ontologyenrichment analysis was performed using the DAVID BioinformaticsResources 6.7 Web site (2003, Dennis et al., Genome Biol 4:P3; 2009,Huang et al., Nat Protoc 4:44-57).

Electrophysiology and Ca2+ Imaging

Patch clamp recordings were performed using the patch clamp technique inthe whole-cell configuration as described previously (29). In brief, GΩseals were achieved using pipettes fashioned from borosilicate glass(WPI) with resistances of 2-3 MΩ after fire polishing. Recordings wereobtained from putative tCardiomyocytes at 25° C. using a resistiveheater system (Warner Instruments). Action potentials were elicitedusing an Axopatch 200B amplifier (Molecular Devices) by injecting 0.4 to0.5-nA pulses at 1-3 Hz with a 0.2- to 0.3-ms duration controlled by aPentium 4-based PC running the pClamp program (v. 9.2; MolecularDevices). Voltage recordings were filtered at 1-2 kHz and digitized at25 kHz using the Digidata 1332A A/D converter (Molecular Devices). Twosolutions were used for current clamp recordings: pipette solution (80mM K+-aspartate, 50 mM KCl, 1 mM MgCl2, 10 mM EGTA, 10 mM Hepes, and 3mM Mg2+ ATP, pH-adjusted to 7.2 with KOH) and bath solution (132 mMNaCl, 4.8 mM KCl, 1.2 mM CaCl2, 2 mM MgCl2, 10 mM Hepes, and 5 mMglucose, pH-adjusted to 7.4 with NaOH). Cytosolic free Ca2+ changes weremonitored as follows. Fluo 4-AM (5 μg/mL in the bath solution)(Invitrogen) was loaded into cultures for 40 min, washed three times inthe bath solution, and de-esterified for 15 min at room temperature.Fluo 4-AM-loaded cells were observed with the Zeiss LSM 710 system at 2-to 3-s intervals (1993, Cheng et al., Science 262:740-744).

The results of the experiments are now described.

Generation of tCardiomyocytes from Fibroblasts Using TranscriptomeTransfer

TIPeR generation of the desired cardiomyocyte phenotype requires anavailable source of cardiomyocyte RNA. Poly-A+ RNA was isolated fromventricular myocytes from adult mice (7-9 wk old) and transfected 2 μgof poly-A+ RNA per 35-mm culture dish into primary mouse embryonicfibroblast cells at passage 3 using cationic lipids. The transfectedcultures were monitored daily for recovery and status. A secondtransfection was performed at varying intervals (3-7 d) after the firsttransfection. These TIPeR cells are referred to as cardio-TIPeR cells.As a control for RNA addition and transfection, fibroblast poly-A+ RNAwas transfected into fibroblast cultures (fibro-TIPeR), using the sametransfection procedure. A cell was considered to be a tCardiomyocyte(cardiomyocyte transcriptome-effected cell) once a cardio-TIPeR cellexpressed any of the cardiomyocyte phenotype from a single-cellphenotyping procedure. Within 2 wk after the first transfection, theoverall morphology of individual cardio-TIPeR cells exhibited amorphology distinct from that of fibroblasts that further discriminatedtCardiomyocytes from nonaffected cardio-TIPeR cells. Compared withfibroblasts, tCardiomyocytes were more 3D under DIC imaging, with anincreased elongated or triangular shape (FIG. 1, DIC images). Asubpopulation of tCardiomyocytes exhibited a triangular shape similar tothat of neonatal cardiomyocytes, whereas other tCardiomyocytes had anelongated rod shape similar to adult cardiomyocytes. The correspondingfibro-TIPeR cells appeared to be larger with a flat morphologyindistinguishable from fibroblasts. Cell morphology analysis measuringthe ratio of maximum cell length to minimum cell width showed that thesubpopulation of cardio-TIPeR cells was distinct from the fibroblastcluster (low length-to-width ratio) and grouped with adult ventricularmyocytes (AVMs) (high length-to-width ratio) (FIG. 1, graph). Thisresult indicates that cardio-TIPeR cells develop morphology distinctfrom that of fibroblasts and fibro-TIPeR cells. The time lapse betweentransfection and the emergence of tCardiomyocyte morphologies suggests arequisite time dependence associated with these morphological changes.The diversity of cell morphologies (neonatal or adultcardiomyocyte-like) implies that a range of gene expression profiles isformed with the introduction and subsequent action of the donortranscriptome. The action of the donor transcriptome occurs in thecontext of the endogenous host transcriptome that varies among hostcells. Although similar phenotypes exist among fibroblasts, the geneexpression profiles are not identical; this applies to AVMs as well.These observations are consistent with the initiation and maintenance ofa cell reprogramming process varying depending on the host cellexpression profile (i.e., relative abundance of gene products).

tCardiomyocyte Expression of Cardiomyocyte Antigenic Markers.

The expression of cardiac markers in tCardiomyocytes was assessed,specifically the cardiac muscle component cardiac troponin I andtranscription factor Nkx2.5, by immunocytochemistry (FIG. 2). Theexpression of cardiac markers was observed at 2 wk posttransfection(FIG. 2, first row) and was stable for the duration of the 8-wkculturing period (FIG. 2, second row). The subcellular distribution ofcardiac markers was similar in AVMs and tCardiomyocytes. Theimmunocytochemistry results were consistent with the cell morphologyresults; both changes were detected at 2 wk after the first transfectionand persisted for more than 8 wk in morphologically distinctive cells.

Reprogramming of Global Gene Expression in tCardiomyocytes

To assess global gene expression changes, the transcriptome profiles ofsingle tCardiomyocytes was compared with single fibroblasts, AVMs, andfibro-TIPeR cells. Single-cell RNA was harvested by capillary-mediatedaspiration. Poly-A+ RNA was amplified using an aRNA amplificationprocedure and used as a probe to screen Affymetrix Mouse Genome 430 2.0microarrays. An informative group of 3,257 probe sets, differentiallyexpressed between fibroblasts and AVMs, was selected and analyzed tocompare gene expression profiles between single tCardiomyocytes andother single cells (FIG. 3A). Hierarchical clustering analysis showedthat three out of five cardio-TIPeR cells (60%) were clustered withAVMs, one cardio-TIPeR cell was located with fibro-TIPeR cells, and theremaining cardio-TIPeR cells were located between the fibroblast groupand the AVM-tCardiomyocyte group. Bootstrap resampling (1,000 times)resulted in the grouping of tCardiomyocytes and AVMs with 93% support.Along with an increase in gene expression for genes traditionallythought to be AVM-enriched, a decrease in fibroblast enriched geneexpression also would be expected. This was seen in the cells that hadtransitioned from the fibroblast phenotype to the tCardiomyocytephenotype (FIG. 3B).

tCardiomyocytes Function as an Electrically Excitable Cell

Because cardiomyocytes are electrically excitable, tCardiomyocytes wereassessed for excitability using the patch clamp technique (FIG. 4A). Itwas found that action potentials could be elicited from tCardiomyocytes,and that these voltage recordings were similar to those obtained fromisolated mature ventricular myocytes (FIG. 4A, first row).tCardiomyocytes had identical resting potentials (−50 mV), with similarupstroke and repolarization patterns, as mature adult ventricularmyocytes, although the peak amplitude of the action potentials fromtCardiomyocytes was slightly lower than that from isolated matureventricular myocytes. Action potentials were recorded in 10 out of 16clamped cardio-TIPeR cells (62.5%). Resting membrane potentials rangedfrom −55 mV to −10 mV, and membranes were instantly depolarized withelectric stimulation. The peak amplitudes and repolarization ratesvaried among tCardiomyocytes. These varying electrophysiologicalcharacteristics suggests that each tCardiomyocyte might have a uniquecomposition of ion channel types and abundances conferring differingelectrical properties (2007, Harrell et al., Physiol Genomics28:273-283). In separate experiments, but in accordance with the actionpotential data, we also observed cytosolic Ca2+ concentration changes intCardiomyocytes (FIG. 4B). tCardiomyocytes demonstrated intracellularlocal Ca2+ oscillations without stimulation (1991, Harootunian et al.,Science 251:75-78; 1993, Cheng et al., Science 262:740-744).

Generation of tCardiomyocytes from Mouse Astrocytes

To examine the dependency of tCardiomyocyte generation on host celltype, AVM poly-A+ RNAs were transfected into primary mouse corticalastrocyte cultures using the identical procedures used for thefibroblast cultures. Cardio-TIPeR astrocyte cultures showed similarprogression as seen in cardio-TIPeR fibroblast cultures. The subset ofcardio-TIPeR astrocytes showed elevated and elongated cell morphologiesat 2 wk after the first transfection, Expression of cardiac troponin Iand Nkx2.5 was observed in astrocyte-generated tCardiomyocytes thatdisplayed elongated cell morphology (FIG. 5A). In addition to theimmunocytochemistry staining, global gene expression changes wereexamined in astrocyte-generated tCardiomyocytes (FIG. 5B). A group ofinformative 1,690 probe sets, differentially expressed between AVMs andtCardiomyocytes versus mock-transfected astrocytes (P<0.01), wasselected and analyzed further. All six AVM mRNA transfected astrocytesingle cells were grouped with single AVMs with 100% bootstrap supportfrom 1,000 resamplings. It should be noted that one of thetCardiomyocytes was clustered within the AVM group, demonstrating thatthis tCardiomyocyte is further along the transdifferentiation pathwaythen some of the other cells by virtue of sharing a more similar globalgene expression profile with single AVMs.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A method of effecting phenotype conversion in acell, said method comprising introducing phenotype-converting nucleicacid of a first cell having a phenotype into a second cell having aphenotype, wherein the phenotype-converting nucleic acid comprises atleast one RNA, wherein the phenotype of said first cell is differentfrom the phenotype of said second cell, wherein the second cell ispre-treated with a transcription inhibitor before it is transfected,wherein said transfected phenotype-converting nucleic acid causes thephenotype of said second cell to change to the phenotype of said firstcell, and wherein said second cell is transfected in vitro or ex vivo.2. The method of claim 1, wherein said phenotype-converting nucleic acidis an mRNA transcriptome.
 3. The method of claim 1, wherein saidphenotype of said first cell differs from said phenotype of said secondcell by one or more of: tissue type, differentiation degree, diseasestate, response to exposure to a toxin, response to exposure to apathogen, and response to exposure to a candidate therapeutic.
 4. Themethod of claim 2, wherein said mRNA transcriptome comprises mRNAtranscripts having an average size between about 1 kb to about 5 kb. 5.The method of claim 2, further comprising introducing said second cellat least a second time with said first cell mRNA transcriptome.
 6. Themethod of claim 1, wherein said first cell is a cardiomyocyte.
 7. Themethod of claim 1, wherein said second cell is a fibroblast.
 8. Themethod of claim 1, wherein said transfection inhibitor is5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB).
 9. The method ofclaim 1, wherein said cell is selected from the group consisting of aeukaryotic cell and a prokaryotic cell.
 10. The method of claim 9,wherein said eukaryotic cell is a non-mammalian cell.
 11. The method ofclaim 9, wherein said eukaryotic cell is a mammalian cell.
 12. Themethod of claim 11, wherein said eukaryotic cell is a human cell. 13.The method of claim 2, wherein said phenotype-converting nucleic acidfurther comprises one or more exogenous nucleic acids selected from thegroup consisting of mRNA, siRNA, miRNA, hnRNA, tRNA, non-coding RNA andcombinations thereof.
 14. The method of claim 1, wherein said phenotypeconversion comprises a change in one or more of gene expression, proteinexpression, immunological markers, morphology, physiology, synthesis ofbioproducts, and membrane lipid composition.
 15. The method of claim 14,wherein phenotype conversion comprises a change in expression of atleast 100 genes.
 16. The method of claim 14, wherein phenotypeconversion comprises up-regulation of genes associated with chromosomalremodeling.
 17. The method of claim 14, wherein at least about 5% ofdifferentially expressed genes in said second cell change expression toa level observed for said first cell.
 18. The method of claim 1, whereinsaid phenotype conversion persists for at least 2 weeks.
 19. The methodof claim 18, wherein said phenotype conversion persists for the lifetimeof the cell.